To develop new methods and probes for protein translocation, we have been developing pharmacologic methods of modulating this process in vivo. We have recently synthesized and characterized a novel small molecule inhibitor of cotranslational protein translocation. This compound (called cotransin) was demonstrated both in vitro and in cultured cells to inihibit the translocation of some, but not other proteins. Remarkably, this substrate-specificity was found to be encoded in the signal sequence. Thus, the sensitivity or resistance to cotransin can be conferred to any protein of interest simply by the choice of signal. The target of cotransin has been identified, and appears to be the Sec61 complex, the central component of the protein translocation channel. These tools and findings now open the way to selectively and potently modulate the translocation of individual substrates in live cells. This approach is now being applied to study the role of protein translocation in various cellular events such a protein aggregation and toxicity, protein degradation, and the cellular response to ER stress. In addition, analogoues of cotransin are now being tested for their effects on a variety of substrates to determine whether specificity of inhibition can be modulated. And finally, cotransin is being used as a tool to dissect the mechanistic basis of signal sequence interaction with the translocon, a decisive step in protein translocation.